A total of 507 endophytic fungal isolates belonging to 82 operational taxonomic units (OTUs) were isolated from fifteen plant species naturally occurring in the Thar Desert, Rajasthan, India. Aspergillus, Alternaria, Chaetomium, Penicillium and
Nigrospora were among the dominant fungal genera. Thermotolerance of the isolates was evaluated by culturing the fungi at 40 °C and 45 °C in shake flasks. Growth of nine OTUs (ACJ-2, ACJ-5, ACL-2, ACT-2, ACT-3, LAS-4, LAS-6, SAP-3 and SAP-6) was unaffected at 40 °C. Of these, six endophytes, namely, ACJ-2, ACJ-5 (Aspergillus flavus), SAP-3 (Aspergillus sp.), SAP-6, LAS-4 (Aspergillus sp.) and LAS-6 (Chaetomium sp.), were tolerant to as high as 45 °C. Rest of the OTUs did not survive culture temperatures beyond 35 °C. We evaluated the ability of one thermotolerant endophyte, LAS-6 (Chaetomium sp.) to confer high temperature tolerance and three OTUs, namely, LAS-4 (Aspergillus sp.), SAP-3 (Aspergillus sp.) and SAP-6 to confer drought tolerance to a rice cultivar, IR-64, at early seedling stage. Seedlings treated with LAS-6 (Chaetomium sp.) showed a higher survival percentage as well as maintained a significantly higher shoot and root growth under high temperature stress compared to seedlings not treated with the fungus. Under drought stress, endophyte treated seedlings maintained a significantly higher root growth compared to untreated seedlings. The identified thermotolerant fungal sources could be potentially useful in alleviating abiotic stress in agriculturally important crops.